An integrated series of biochemical, biophysical, and combinatorial experiments is designed to answer three fundamental questions concerning the hairpin ribozyme: First, how does the RNA assemble into a catalytic structure? Second, what is the structure of the ribozyme-substrate complex? Third, how does this structure result in the acquisition of catalytic activity? The hairpin ribozyme is well suited to such experiments, since the molecule is small enough that comprehensive studies can be undertaken, yet complex enough that significant conformational changes occur during the catalytic cycle. The hairpin differs in several important respects from the only two RNA enzymes that have been more extensively studied, the hammerhead ribozyme and group I introns. Specific Aims are: (1) Map the tertiary structure of the ribozyme-substrate complex; (2) Determine the mechanisms through which metal ions and inhibitors modulate catalytic activity; (3) Define the folding pathway and determine the requirements, kinetics, and thermodynamics of folding and unfolding; (4) Determine the tertiary structure of the ribozyme- substrate complex at high resolution; and (5) Integrate results to develop and test models of the catalytic mechanism. The importance of this work includes advancement of our understanding of RNA structure and how it leads to catalytic activity, the role of catalytic RNA and RNP in biological reactions, and the application of our understanding of catalytic RNA and ribozyme engineering to improving heath, through identifying gene function in genomic investigations and the development of selective ribozyme-based therapeutics for genetic and viral diseases.